Cell chirality reversal through tilted balance between polymerization of radial fibers and clockwise-swirling of transverse arc

doi:10.21203/rs.3.rs-1308149/v1

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Cell chirality reversal through tilted balance between polymerization of radial fibers and clockwise-swirling of transverse arc

https://doi.org/10.21203/rs.3.rs-1308149/v1

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Cell chirality is an intrinsic property shown as biased cell rotation or orientation. While the righthanded helix of actin is known essential, it is intriguing of how single form of molecular handedness can be manifested into diverse and even mirrored forms of cell chirality. Here, we found that cell nucleus rotates with clockwise (CW) bias with smaller projected area but reverses to anticlockwise (ACW) bias when cell spreading increases. Actin analysis suggests that polymerization of righthanded radial fibers accounts for the ACW bias, while the CW bias is driven by the retrograde flow of CW-swirling transverse arc, which originates from tethered myosin II that simultaneously connect radial fibers and transverse arc. Thus, by tilting the balance between these two classes of actin fibers via cell spreading area or the main factors of actin, i.e., myosin II, α-actinin-1, Tpm4, and mDia2, the single form of actin helix can be unfolded into cell chirality with opposite bias. This finding suggests a mechanistic insight of cell chirality reversal, providing new perspective in mechanobiology and tissue formation.

Development of animal’s tissue or organ requires establishment of left-right (LR) asymmetry ¹, which includes LR body axis for visceral distribution ^2,3 and chiral morphogenesis in tissue/organ architecture ^4–7. However, how LR asymmetry spontaneously emerges remains largely unknown. Cell chirality, a mechanical behavior such as tilted cell orientation or clockwise/anticlockwise-biased cell rotation, has been postulated as the origin of LR asymmetry in tissue level ^1,8. For example, blastomeres in C. elegans embryos exhibit a chiral skew event that results in an uneven distribution of cells ^2,9. In later stage of embryonic development, such chiral mechanics caused a collective motion in epithelial morphogenesis ^6,10,11, which eventually directs the formation of genitalia ¹¹ and axial torsion of a hindgut ^6,10. In addition to embryonic development, footprints of cell chirality can be still seen by their ability of generating cellular torque ^9,12, migration with LR bias ^13–16, or forming specific alignment in the multicellular level ^14,17,18. Through cell-cell communication, the chiral behavior causes LR-biased cell assembly of multicellular structure ^14,19 and regulates permeability of intercellular junctions ²⁰.

Importantly, actomyosin cytoskeleton was found important in cell chirality. Using 3D Riesz transform-differential interference contrast, it revealed a chiral motility of filopodia in neuronal growth cone and amoeba ²¹. For cells on micropatterns, the expression of chirality depends on the actomyosin activity at the micropattern boundaries ^14,22. Essentially, actomyosin activity was also reported important in vivo, since the use of its inhibitors may abolish or even reverse the LR features of tissue morphogenesis ^4,6. Such notion was further supported by a recent report which directly elucidated a chiral alignment of actin filament ²². Specifically, when cultured on micropatterned circles, radial actin fibers that point from cell periphery to nucleus are unidirectionally tilted, forming a chiral swirling pattern of actin filament. With such chiral patterns, the intracellular actin filament serves as the main driver of this chiral torque and rotation ^12,23,24.

It is believed that that a helical motion of actin filament may underlie the cell chirality ^21,22 because actin filament is only formed with righthanded double helix. However, it has been intriguing and puzzling of how single form of molecular chirality can be manifested in diverse chirality at cellular level that is even mirrored from one cell type to another ^12,17. Recently, righthanded axial spinning of radial fiber during polymerization has been proposed as the underlying mechanism for the anticlockwise (ACW) chiral pattern of actin filament ²⁵. In addition, such ACW chirality can be reversed through overexpression of α-actinin-1 that cross-links the actin filament or modulation of actin polymerization and depolymerization ^19,25−27. However, as the molecular handedness of actin filament remains unchanged, the emergence of clockwise (CW) chirality by either cross-linking or altered polymerization of righthanded actin filament remains vague and unresolved.

Here, we report the reversal of cell chirality through switching dominance between two classes of actin fibers. We first observed reversal of nucleus chiral rotation from CW-biased to anticlockwise ACW-biased during initial cell attachment. Using micropatterning, the chirality reversal appears to be dependent on the increase of cell projection area and is universally seen in different cell types. Analysis of actin distribution with small-molecule drugs found that polymerization of radial fibers arising from their barbed end near the leading edge is essential for the ACW chirality, and they are suppressed on small islands. In contrast, time-lapse microscopy shows that CW chirality originates from a CW swirling of transverse arc. During the retrograde flow, it approaches toward the nucleus, eventually causing a CW rotational wrapping around the nucleus. Importantly, silencing of tropomyosin 4 (Tpm4) detaches myosin II from radial fibers and increases ACW chirality, suggesting that the attachment of lefthanded-walking myosin II may drive a lefthanded twirling of radial fibers which eventually leads to CW swirling of transverse arc. Thus, by tilting the balance between the two classes of actin stress fibers by means of adjusting cell spreading area or the main components of actin fibers, e.g., myosin II, α-actinin, Tpm4, the chirality is reversed. Furthermore, based on the induced ACW chirality through silencing of mDia2, the upstream factor of transverse arc formation, osteogenesis is enhanced, suggesting the role of the early committed chirality in guiding the lineage specification. This work reveals a general, previously unidentified mechanistic understanding of chirality reversal, providing a unique opportunity unveiling the role of cell chirality in controlling tissue morphogenesis.

Cell Chirality is Reversed from CW to ACW Bias during Cell Attachment.

Attachment to cell-adherent substrate is the prerequisite for expression of cell chirality ^14,17. We first took time-lapse microscopy to record cell spreading and nuclei rotation during spreading of human foreskin fibroblast (HFF-1) on culture dish. Cells spread from circular shape to its natural shape from 0 min to 240 min (Fig. 1a and Supplementary Movie 1). To assess the cell chirality, cell nuclei were tracked using the binarized nuclei images to record the trajectory of nuclei movement with 5 min interval (Fig. 1c and 1d). After recording, the chirality is quantified as the probability of CW and ACW rotation within every 15 minutes for all cells. Our results reveal that nucleus rotation begins with CW bias after initial attachment but is reversed to ACW-biased with time (Fig. 1e). Noticeably, during the temporal change of cell chirality, the cell projected area increases (Fig. 1f) while the circularity decreases as cells are approaching to its natural shape (Fig. 1g, h), suggesting a possible dependence of CW/ACW bias to them. Because cells with non-circular shape would inhibit expression of chiral rotation of cells,¹² the increased projection area is more likely the reason of chirality reversal. To clarify it, the probability of ACW/CW rotation was further analyzed against the averaged projected area. For circularity greater than 0.7, the reversal of chirality was found (Fig. 1i). In contrast, it is not as apparent for cell circularity below 0.7 (Fig. 1j). Together, the results suggest that the chiral nucleus rotation may require circular cell shape and can be revered from CW to ACW with increase of cell projected area.

Cell Chirality is Reversed from CW to ACW with Increment of Micro-island Area.

To provide more direct evidence of the role of cell projected area to the chirality reversal, we applied arrays of fibronectin micro-islands with different circular areas, surrounding by cell-repellent Pluronic coating (Fig. 2a). Time-lapse measurement of nucleus rotation of HFF-1 fibroblasts was again used to record nucleus rotation on micro-islands. Interestingly, when cells spread on 500 µm² pattern, the nucleus rotation is neutral (Fig. 2b). Starting from 750 µm², nucleus rotation becomes CW-biased, i.e., 45.83 ± 1.96% ACW with p-value < 0.01, which is consistent with the chiral bias of cell during initiate spreading. From 1000 µm² to 2500 µm², the chiral nucleus rotation is changed to neutral and then reversed to ACW-biased, i.e., 63.43 ± 2.90% ACW with p-value < 0.0001 on 2500 µm² of micro-islands. Thus, the results on micro-islands provide a direct and consistent evidence showing cell projected area as the key factor for chirality reversal. In contrast, for cells on other non-circular shapes, e.g., triangle, square, and rectangle with area of 750 µm² or 2500 µm², the reversal of cell chirality from small to large island is not seen, indicating circular islands as a better platform for exhibition of area-dependent chirality reversal (Supplementary Fig. 1). Furthermore, to test the universality of such phenomenon, we have analyzed the nucleus rotation on two other cell types (Fig. 2c-d), C2C12 mouse myoblasts and human mesenchymal stem cells (hMSCs). They all show the same dependence of chirality reversal based on cell spreading area, demonstrating the universality of this relationship.

Actin Distribution upon Drug Treatment Reveals the Role of Actin Polymerization for ACW Chirality.

To investigate the mechanism, we next examined actin cytoskeleton on such islands. We have previously found that actomyosin cytoskeleton may function as a built-in machinery underlying the cell chirality ^14,17. At the micropattern boundaries, actin filament even arises as a chiral swirling pattern composed of transverse arc and radial fibers ^28,29 when cultured on micropatterned circles ²². By staining actin filaments for HFF-1 fibroblasts on large islands (2500 µm²), actin forms a swirl pattern of radial fibers in conjunction with transverse arc and can be seen as a ring shape after image stacking (Fig. 3a). Using automated image processing to analyze the tilting angle of actin filament with respect to the radial axis of each cell ²³, a ACW bias was found (Fig. 3b), which is consistent with previous findings ²² and the nucleus rotation mentioned above. Interestingly, with nucleus removed by treatment of cytochalasin D followed by centrifugation and recovery in the growth medium, the ACW bias of actin filament is maintained, suggesting actin as the main driver of chiral nucleus rotation (Supplementary Fig. 2). Surprisingly, while CW rotation is observed for cells on small islands (750 µm²), only ventral fibers and transverse arc remain, and radial fiber is rarely observed (Fig. 3c). After image stacking, the actin ring is replaced by accumulation of actin at cell center. Together, it suggests that actin is the major driver of cell chirality, but not necessarily to be solely dependent on the presence of radial fibers.

Such finding is unexpected as previous findings suggested that the chiral swirling of actin filament was initiated from a possible axial rotation of radial fibers during polymerization ²². To further investigate it, we applied a series of cytoskeleton-related inhibitors on HFF-1 fibroblasts on two representative micro-islands, 750 µm² as small island inducing CW bias and 2500 µm² as a large island inducing ACW bias. We have identified A23187 Calcium Ionophore (A23) that stimulates perinuclear actin formation ³⁰, the inhibitor of F-actin polymerization Latrunculin A (LatA) ³¹, RAC1 inhibitor NSC23677 (NSC) ³², formin FH2 domain inhibitor SMIFH2 ^33,34, and activator of actin-based complexes Lysophosphatidic acid (LPA) ³⁵. On large islands (2500 µm²), the original ACW chirality (63.43 ± 2.90% ACW) remains unchanged with A23 treatment (Fig. 3d) and becomes neutralized by LPA treatment and even more ACW-biased with NSC and SMIFH2 (Supplementary Fig. 3a). In contrast, treatment of LatA could reverse the chirality to CW bias (45.58 ± 2.15% ACW, Fig. 3d). On the other hand, the CW bias on small islands (750 µm²) is unaffected by the treatment of LatA (Fig. 3e), but is neutralized by NSC, SMIFH2 and LPA (Supplementary Fig. 3b). More interestingly, A23 reverses the nucleus rotation from its original 45.83 ± 1.96% ACW to 54.00 ± 1.60% ACW bias (Fig. 3e). From the above results, treatment of A23 and LatA appear to be effective to reverse the induced CW/ACW bias on small/large island, respectively.

We next analyzed the actin distribution of HFF-1 fibroblasts in response to the A23 and LatA treatment. Comparing to untreated cells, actin is lessened around the nucleus with A23 treatment, but more ventral stress fibers across cell nucleus can be seen with LatA treatment (Fig. 3f). To statistically illustrate actin distribution upon treatment, we applied image stacking. A23 treatment pushes the actin around the nucleus toward the cell edge (Fig. 3g), while the LatA treatment, to the opposite, brings actin closer to the center, leading to more equalized actin distribution. For cells on small islands, similar effects are seen, and more interestingly, radial fibers are restored with A23 treatment (Fig. 3h, i). To highlight the difference, we used differential heat map by subtracting the stacked actin image between treated and untreated cases. Clearly, A23 concentrates actin at the cell edge but lessen it at the center (Fig. 3j) and a distinct peak of actin intensity appears immediately adjacent to the cell edge (dash line) (Fig. 3k). In contrast, for LatA treatment, actin is concentrated around the nucleus while the peripheral ring of actin is lessened than that of untreated control (Fig. 3j-k). Same effects are also seen in cells on small islands (Fig. 3l-m). Because polymerization of radial actin fibers arises from nucleation of actin filament at their barbed end near the leading edge, such observation suggests an increased polymerization of radial fibers upon A23 treatment which may account for the ACW chiral rotation, especially for cells on smaller islands where their original CW rotation can be reverted to ACW rotation upon restoration of radial fibers. On the other hand, as an inhibitor of F-actin polymerization, LatA appears to suppress the polymerization of radial fibers at cell edge, which may underlie the induced CW nucleus rotation of cells on large islands with LatA treatment.

CW-Swirling of Transverse Arc Acts as the Main Driver for CW Chirality.

How does actin polymerization near the cell edge affect the cell chirality? To elucidate the mechanism, we conducted transfection of LifeAct in HFF-1 fibroblasts. Time-lapse video microscopy shows centripetal-growing radial fibers originate from the cell periphery (Fig. 4a and Supplementary Movie 2, 3). With increase of length, the radial fibers start to tilt rightward accompanying with retrograde flow along the right-tilted growth direction of radial fibers which eventually drives an overall ACW rotation of entire cytoskeleton around cell nucleus. As such, the actin filament forms a chiral swirling pattern with strong coherence of nucleus rotation. Such inward growing filament with rightward turning is consistent with the literature ^21,22 and can be explained by the righthanded double helical structures of actin ³⁶. As the barbed ends of actin filament is capped and tethered by formin, incorporation of new actin monomer to the righthanded double helix would cause the other free end of actin filament growing with righthanded axial spinning (Supplementary Movie 4) ²². In contrast, for cells on small islands, the nucleus rotation is solely driven by the retrograde flow of transverse arc without apparent radial fibers (Supplementary Movie 5).

On the other hand, for cells with LatA treatment, while some radial fibers are seen, such rightward turning is absent (Fig. 4b and Supplementary Movie 6). Instead, we noticed a very organized CW swirling of transverse arc that approaches to the nucleus during the retrograde flow, eventually becoming a CW-bias swirling wrapping that causes the nucleus to commit to CW rotation (Fig. 4b and Supplementary Movie 6). Such active transverse arc tends to connect radial fibers such that ventral stress fibers across cell nucleus are formed. As a result, for cells on large island with LatA, more actin is concentrated around the nucleus (Fig. 3j). In some cases, the CW-bias of nucleus rotation occurs with this CW-bias swirling wrapping of transverse arc without distinct, bundled radial fibers (Supplementary Movie 7).

To further prove the role of retrograde flow of transverse arc in CW rotation, we perturbed the major components of transverse arc. Transverse arc are curved actomyosin bundles ²⁸ formed end-to-end annealing of α-actinin and myosin II bundles in a periodic pattern ²⁹. Different from radial fibers, transverse arc are contractile because the incorporation of myosin II bundles which, together with polymerization of radial fibers, drives the retrograde flow of transverse arc ³⁷. Thus, perturbation of α-actinin and myosin II would impose direct influence on the transverse arc. We first applied overexpression of α-actinin, which could promote the bundling of radial fibers and restrict their axial spinning. Meanwhile, it would also promote the assemble of actin filament during transverse arc formation. Our results show an enhanced CW rotation with increasing α-actinin expression (Fig. 4c-d). Next. we applied Y27632, the inhibitor the Rho-associated kinase (ROCK) to down-regulate the myosin light chain phosphorylation ³⁸. Interestingly, myosin II highly enriched in transverse arc in untreated cells is greatly reduced after inhibition by Y27632 (Fig. 4e). More importantly, it enhances the ACW nucleus rotation for cells on both large and small islands, which supports the role of transverse arc in CW bias of nucleus rotation (Fig. 4f). Together, our results suggest the right-turning of centripetal-growing radial fiber drives the ACW rotation, while the retrograde flow of CW swirling of transverse arc is responsible to the CW-biased nucleus rotation.

While the ACW chirality has been explained by the righthanded axial spinning of radial fiber during polymerization ²² (Fig. 5a and Supplementary Movie 4), how such single form of molecular handedness can be unfolded to CW swirling of transverse arc remains intriguingly and unclear. We noticed that myosin II, while presents in the region of transverse arc mostly, does colocalize with radial fibers (Fig. 4e and the zoom-in image). This finding is consistent with previous findings that tropomyosin localizes to radial fibers and coincides with myosin II incorporation ³⁹. Thus, myosin II may be involved in the connection between radial fibers and transverse arc. Interestingly, myosin II and V have been shown as lefthanded spiral motor on the righthanded actin helix ^36,40 (Fig. 5b and Supplementary Movie 8). During the actin gliding, the lefthanded translocation of myosin II could, on the other hand, cause lefthanded twirling of an actin filament about its axis ⁴⁰. Similar phenomenon of lefthanded twirling of actin has been reported in the retraction of filopodia where its right-screw rotation can later lead to righthanded clockwise cell migration ²¹. Thus, when transverse arc is dominant, the attachment of tethered myosin II, because it simultaneously attaches to fragment of crosslinked arc filaments, could plausibly cause a lefthanded twirling, or axial spinning of radial fibers growing inward (Fig. 5c and Supplementary Movie 9). As a result, opposite to the rightward tilting of radial fibers based on the righthanded spinning during actin polymerization, a short radial actin filament connecting to transverse arc precursors could exhibit a lefthanded spinning and tilted leftward. Eventually, the radial fibers tilting with either rightward or leftward direction would be in conjunction to retrograde flow of transverse arc, therefore causing the overall ACW or CW rotation of nucleus (Fig. 5d, e).

To further investigate the role of myosin II in the formation of CW swirling of transverse, we applied siRNA gene silencing to knockdown myosin binding protein tropomyosin. Tropomyosin has been reported with isoform-specific localization along radial fibers ³⁹. Among the isoforms, Tpm4 on radial fibers is particularly important for recruitment for myosin II as its presence on radial fibers coincides with the incorporation of myosin II. We first tried to observe the distribution of Tpm4 in untreated cells. Tpm4 decorates the radial and ventral actin fibers (Fig. 5f). Interestingly, in the Tpm4 silencing cell, myosin IIa on radial fibers is less condensed (Fig. 5g), indicating the disrupted attachment of myosin II. Consistently, the actin distribution shows a suppression of transverse arc. Noticeably, the transverse arc is more apparent near the periphery of cells but not forming the distinct actin ring as seen in cells of normal condition (Fig. 5h and Fig. 3a). Such result collectively suggests the lost connection of transverse arc precursors to radial fibers such that they fail to move inward through the retrograde flow. More critically, the nucleus rotation of HFF-1 with Tpm4 silencing exhibited much greater ACW chirality both on large patterns (from 63.43 ± 2.90% ACW to 81.64 ± 2.79% ACW) and small (from 45.83 ± 1.96% ACW to 61.37± 3.09% ACW) (Fig. 5i). In contrast, Tpm1, Tpm2 and Tpm3 knockdown did not show noticeable effect on chirality (Supplementary Fig. 5). Based on the above results, we can conclude that the attachment of myosin II on radial fibers via Tpm4 is important for conducing CW swirling of transverse arc.

Endogenous Factor Regulates Chirality Reversal to Differentiation.

We next explore the existence of endogenous factor that could intervene the balance of radial fibers and transverse arc and reverse the cell chirality in HFF-1 fibroblasts. Compared to mDia1 that is mostly responsible for the actin polymerization at barbed end ⁴¹, mDia2 promotes the nucleation of Tpm4-decorated actin filaments, which later recruit myosin II before assembling to α-actinin-crosslinked actin filaments ⁴². Thus, mDia2 may more likely be the upstream factor to tilt the balance between radial fibers and transverse arc. HFF-1 cells with siRNA interfering mDia2 induces an overall reduction of mDia2 expression (Supplementary Fig. 6a). Very importantly, mDia2 silencing reduces the myosin IIa on transverse arc and depletes the co-localization of myosin IIa on radial fibers (Fig. 6a), indicating that mDia2 silencing down-regulates not only the recruitment of myosin II to the assembly of actomyosin bundles but also the connection between radial fibers and transverse arc. With such effect, the cell chirality is tilted to the dominance of radial fibers, which is shown by more ACW bias on both large islands (from 63.43 ± 2.90% ACW to 67.79 ± 3.04% ACW) and small islands (from 45.83 ± 1.96% ACW to 50.88 ± 1.67% ACW) (Fig. 6b). Interestingly, such enhanced ACW bias together with the actin distribution (Supplementary Fig. 6b-c) is similar to the results of formin FH2 domain inhibition by SMIFH2 (Supplementary Fig. 4). To further examine the role of mDia2 in CW rotation, we used transfection of GFP-mDia2 for over expression assay. Interestingly, overexpression of mDia2 causes an overall increase of mDia2 (Supplementary Fig. 6a) and enhanced CW bias of cells on both sizes of islands (Fig. 6c). For the actin distribution, the dominance of transverse arc is seen (Supplementary Fig. 6d-e), and an increase of actin at cell center (Supplementary Fig. 6e) resembling the effect of LatA treatment is observed (Fig. 3g, j). Together, the results suggest that mDia2 as one of the upstream endogenous factors to tilt the balance between radial fibers and transverse arc.

We next explored the potential regulation of cell differentiation by adjusting the cell chirality. Previously we have shown that hMSCs would early committed to a CW chirality with adipogenic induction, which may possibly serve as the mechanical precursor to engage the cell phenotype toward the adipogenic lineage ²³. To explore whether the expression of mDia2 can guide the lineage specification, we applied mDia2 silencing to hMSCs (24 hours of transfection and 12 hours of recovery in growth media) before adipogenic and osteogenic dual induction and count the lineage specification by Oil Red O staining (for adipocytes) and Fast Blue staining (for osteoblasts) (Fig. 6d). Interestingly, the transient silencing conducted before induction can still result in an increased ACW bias (from 49.54 ± 4.68% ACW to 56.75 ± 2.82% ACW) after 6 days of dual induction (Fig. 6e), suggesting a long-term effect of mDia2 silencing. More importantly, mDia2 silencing causes a reduced percentage of adipocytes (Fig. 6f), which is consistent with literature that early committed CW bias would induce adipogenesis ⁴³, suggesting the role of cell chirality in guiding the lineage specification.

In this study, we first observed chiral reversal from CW to ACW bias during cell spreading. Using micropatterning, the CW bias of nucleus rotation on smaller islands is reversed to ACW bias with increased area of micro-islands, suggesting the dependence of cell projected area. Examination of actin distribution indicates the enhanced peripheral actin at the cell edge when nucleus rotates with ACW bias, suggesting the role of enhanced actin polymerization at their barbed end near the leading edge that leads to growing radial fiber with right turning. In contrast, the CW bias is associated with concentrated actin around the nucleus. Using time-lapse video microscopy, we found that the CW swirling of transverse arc during the retrograde flow, which creates CW-bias swirling wrapping causing the nucleus to commit to the CW-biased rotation. Further study indicates that the attachment of tethered, lefthanded-walking myosin II on transverse arc and radial fibers could plausibly cause a lefthanded twirling of radial fibers growing inward. Eventually, the leftward or rightward tilting of radial fibers depending on the dominance of polymerization of radial fibers or CW swirling of transverse arc is in conjunction to retrograde flow which causes the overall ACW or CW rotation of nucleus (Fig. 5d, e). Supporting this idea, we do observe that radial fibers in untreated cells could initially tilt rightward or leftward possibly depending on the initial dominance of righthanded actin polymerization or lefthanded myosin II power strokes, before commitment to the final, overall rotation of cytoskeleton (zoom-in image in Fig. 4a and Supplementary Movie 3). In contrast, such sharp change of radial fiber orientation is less seen in cells with treatment of LatA, Y27632, or Tpm4 silencing as the dominance of transverse arc and radial fibers are already enforced (zoom-in image in Fig. 4b, e and Fig. 5h).

The key underlying the formation of CW swirling of transverse arc is the lefthanded-walking myosin II that simultaneously connect to radial fibers and transverse arc. The decoration of Tpm4 on radial fibers and its role in recruiting and colocalizing myosin II are consistent with the literature ³⁹. However, as the growth of radial fibers is known to be driven by actin polymerization at their barbed end, the exact mechanism by which Tpm4 is localized on the radial fibers has been elusive. In our result, mDia2 silencing has greatly reduced the assembly of myosin II bundles to actin filament in transverse arc as well as their attachment to radial fibers (Fig. 6a). Importantly, tropomyosin-decorated actin filaments are typically nucleated by mDia2, which then recruits myosin II and anneals endwise with α-actinin-crosslinked actin filaments, eventually condenses to myosin II-containing transverse arc ³⁹. As annealing of α-actinin is also present in radial fibers, we believe that tropomyosin-decorated actin filaments nucleated by mDia2 may also assemble to radial fibers through α-actinin-crosslinking. Thus, the Tpm4 decoration can then recruit myosin II shown as spots located along the radial fibers, which serves as the connection points between radial fibers and arc precursors.

Studies of upstream, endogenous factor indicates the role of mDia2 in manipulating actin structure and intervene the balance of radial fibers and transverse arc. Moreover, silencing of mDia2 before induction of hMSCs could also stimulate an enhanced ACW rotation of hMSCs observed even after 6 days of induction, and guide the lineage commitment toward osteogenic differentiation, suggesting role mDia2 in modulating lineage specification. While cell chirality has been reported with diverse forms, the factors that breaks the balance of radial fiber and transverse arc for chirality reversal and its potential function in lineage specification is revealed here for the first time to our knowledge.

Our results suggest an increased peripheral actin near the cell edge upon treatment of calcium ionophore A23. Previously, A23 has been shown to reproduce the reorganization of the perinuclear actin probably triggered by an increase in cytosolic Ca²⁺ concentration ³⁰. While not entirely understood, it was been hypothesized that increased actin polymerization may be a result of increase of G-actin after Ca²⁺-dependent disassembly. Thus, the G-actin can promote of actin nucleation by mDia1⁴⁴, which subsequently leads to polymerization of radial fibers. To the opposite, treatment of LatA inhibits F-actin polymerization by forming 1 to 1 molar complex with G-actin ³¹. As the peripheral actin is indeed reduced, our results collectively suggest the essential role G-actin in regulation of actin polymerization at cell edge that accounts for the growth of radial fibers.

The retrograde flow of transverse arc is consistently higher than the growth rate of radial fibers, suggesting the involvement of myosin II-dependent contractility of the transverse arc ²². Thus, our results of Y27632 treatment should not only disrupt the connection between radial fibers and transverse arc, as mentioned above, but also reduce the centripetal motion of transverse arc, therefore tilting balance toward radial fibers and enhancing the ACW chirality. On the other hand, LatA treatment does not reduce the retrograde flow of transverse arc but even slightly increase it ²², which would further enhance the influence of CW-swirling wrapping around the nucleus. While the mechanism of remains unclear, the velocity of retrograde flow may account for the CW chirality of cells.

In summary, using nucleus rotation, our results indicate the main drivers for ACW and CW chirality, i.e., righthanded axial spinning of growing radial fibers for ACW bias and centripetal wrapping of CW spiral transverse arc for CW bias. By tilting the balance between these two classes of actin fibers by means of adjusting cell spreading area or regulating the main components of actin fibers, e.g., myosin II, α-actinin, and Tpm4, the cell chirality can be enhanced or reversed. Moreover, silencing of mDia2, the upstream factor of transverse arc formation, can induce ACW bias during induction and eventually lead to increased percentage of osteogenesis. This finding provides a general mechanism of chirality reversal, which has broad implications applicable to different cell types and understanding of tissue morphogenesis.

Cell Culture. HFF-1 human foreskin fibroblasts (SCRC-1041™, ATCC) were cultured in Dulbecco's Modified Eagle Medium (4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate and 1.5 g/L sodium bicarbonate, Life Technologies) supplemented with 15% fetal bovine serum and 1% penicillin–streptomycin (Life Technologies). Only from passage 2 to 10 would be used for experiment. C2C12 mouse myoblast (CRL-1772™, ATCC) was thawed in P8 and used at P9. They were cultured in Dulbecco’s Modified Eagle’s Medium (4 mM L-glutamine, 4500 mg/L glucose and 3.7 g/L sodium bicarbonate, Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Life Technologies). Human mesenchymal stem cells (hMSCs, PT-2501, Lonza) was cultured in Dulbecco’s Modified Eagle’s Medium (Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Life Technologies). To examine the role of actin in nucleus rotation, cells were treated with 50 nM of Latrunculin A (Sigma- Aldrich), 100 µM of NSC23766 (Sigma- Aldrich), 5 µM of SMIFH2 (Sigma- Aldrich), 2 µM of A23187 (J&K Scientific), 1 mM Lysophosphatidic acid (Sigma-Aldrich), or 10 µM Y27632 (Alexis).

Nucleus Rotation on Culture Dish. After confluence, the nuclei of HFF-1 cells were stained by applying 2 µg/ml H33342 trihydrochloride (Sigma-Aldrich) in medium for 30 min. Cells were suspended and reseeded onto a culture dish at density of 5000 cells per cm² and incubated in 37°C incubator for 15 min for attachment. After removing the non-attached cells and replenishing with fresh medium, the dish was placed in a live cell incubator on microscope stage (Chamlide^TM TC incubator system, Live Cell Instrument) with 100% humidity and 5% CO₂ in 37˚C. To record the nucleus rotation, fluorescence images of nucleus and phase contrast images were acquired by Nikon Eclipse Ti-E microscope with 5 min interval for 4 h. Cell tracking and labeling was performed based on the shortest displacement by comparing the locational information of each cell at the current image and previous time interval, as shown by the trajectory of cell location for 4 h after cell attachment to culture dish (Fig. 1b). Automated image segmentation was used to measure the angular velocity of nucleus. In brief, after image binarization, the contour of cell nucleus was fit by an oval shape to identify its long axis. By calculating the transient angular velocity of the long axis within the time interval, angular velocity was calculated by the angle difference of the nucleus at two consecutive images, which gives the transient angular velocity of each cell of each 5 min interval for total 4 hour and 30 min. By grouping measurement of all cells within every 15 min, the probability of ACW or CW rotation was determined. Afterward, the difference between the ACW/CW percentages was compared based the projection area and circularity calculated based on cell measured in Nikon NIS-Elements.

Micropatterning. Photolithography was used to accomplish a series of cell adherent islands with varied size. Glass slides were treated with piranha solution (sulfuric acid to hydrogen peroxide = 3:1, 100˚C) for an hour. After rinsing and drying, the slides were treated with hexamethyldislazane (HMDS, Sigma- Aldrich) coating in vapor phase for 5 min. AZ5214 photoresist (PR, AZ Electronic Materials, Luxembourg) was spin-coated on substrates at 3000 rpm and baked at 95°C for 2 min. The photoresist was undergone UV exposure and developed for using developer (AZ400k: deionized water 1:3) to imprint the desire pattern, followed by plasma treatment to remove the remaining photoresist on the pattern (800 mTorr at 30W). Next, the pattern was coated with fibronectin solution (20 µg/ml, Life Technology) for an hour. Afterward, the remaining photoresist was removed by rinsing in absolute ethanol for 3 times, 5 min each, and dried before use. Finally, the substrates were treated with 1% Pluronic F127 for 50 min before cell seeding.

Nucleus Rotation on Circular Micropatterns. HFF-1 fibroblasts and C2C12 myoblasts were seeded at density of 4000 cells/cm² on circular patterned glass with different size ranging from 500 to 2500 um², while hMSCs were seeded at density of 1000-2000 cells/cm² on pattern size ranged from 1000 to 5000 um². After incubation for 30 min, cells were replenished with fresh growth medium for 3 h. Cells were treated with 2 µg/mL bisBenzimide H33342 trihydrochloride (Sigma-Aldrich) for 30 min and imaged by Nikon Eclipse Ti-E microscope at every 10 min interval for 2-3 h. The automated image segmentation was used for measuring the nucleus rotation as aforementioned. The percentages of ACW or CW transient rotation of each cell was determined within the entire duration and averaged among many cells

Analysis of Fiber Orientation on Circular Pattern. The quantitative analysis of actin fiber orientation was carried out in Matlab as described previously ²³. In brief, adaptive threshold and Gaussian filter was applied on cropped actin image to generate a binarized image of actin. Central region of actin was removed to highlight the radial fibers and transverse arc. The core skeleton of actin filament was then obtained by removing lines with shorter than 8 pixels. Skeleton was classified as straight line (aspect ratio of surrounding ellipse of the line is greater than 4) or curve lines. For straight skeleton, its orientation is obtained by the acute angle difference between the long axis of ellipse and the tangential direction to the center. For curve skeleton, the orientation is calculated by the location of pixel on the curve line determined by the 3-point spline method and the tangential line to the center. Orientation of actin is defined as ACW when orientated from 0° to 90° and CW when orientated from -90° to 0°.

Plasmid Transfection. HFF-1 fibroblasts were seeded one night before the transfection (100,000 cells in 35 mm dish). We used the DNA plasmid, mRuby-Lifeact, a gift from Dr. Cheng-Han Yu from University of Hong Kong, pCDNA_Lifeact-GFP_NLS-mCherry was a gift from Olivier Pertz (Addgene plasmid # 69058), pEFmEGFP-mDia2, a gift from Arthur Alberts (Addgene plasmid # 25407) and pEGFP-N1 alpha-actinin 1, a gift from Carol Otey (Addgene plasmid # 11908). The OPTIMEM medium (ThermoFisher) and Lipofectamine 3000 Assay (ThermoFisher) were used in transfection. According to manufacturer suggestion, 500 ng of DNA plasmid was put in OPTIMEM medium. Following the addition of P3000 Reagent and Lipofectamine 3000 Reagent, the mixture was incubated for 30 min to form lipofectamine complex before applying to cells. Cells were incubated with transfection mixture overnight, followed by replenishing with fresh medium for recovery. Next, the transfected cell was reseeded on micropatterned surface at density of 4,000 cells/cm². After incubation of 30 min for cell to spread fully, fresh medium was replenished before imaging.

Gene Silencing. The siRNA was from Invitrogen Silencer® Pre-designed siRNA, to deplete Tropomyosins, Tpm1 (AM51331), Tpm2 (AM51331), Tpm3 (AM16708), Tpm4 (AM16708) and mDia2 (AM16708) (Supplementary Table. 1). Transfection on HFF-1 was based on Lipofectamine 3000 Reagent procedure same as mentioned above with final concentration of siRNA at 75 nM. After 24 h of transfection, fresh medium would be replenished, and experiment (nucleus rotation and fluorescence imaging) was done on day 3 after the addition of transfection complex. For hMSCs, transfection of mDia2 siRNA was carried out by Lipofectamine RNAiMax. Cell was seeded in 9,500 cells per cm² density prior the day of transfection. Lipofectamine RNAiMax was diluted and incubated with siRNA for 15 min before supplementing into cells to make the concentration of siRNA at 50 nM for 24 hours of transfection, followed by 12 h of recovery in growth media before induction. Recovered from transfection on Day 6, cell would be treated with 2 µg/mL bisBenzimide H33342 trihydrochloride for 30 min, before seeding on micropatterned circular islands. After incubation of 30 min for cell to spread fully, fresh medium was replenished before imaging.

Fluorescent Staining. HFF-1 fibroblast was seeded on circular islands with size of 750 µm² and 2500 µm² at density of 4,000 cells/cm² for 30 min. Next, they were replenished with growth medium for 6 h. The cell was then treated with 4% of paraformaldehyde (PFA) for 15 min, 0.2% Triton X-100 for 10 min, and Image-iT^TM FX (Thermo Fisher Scientific) signal enhancer for 30 min. Next, depending on the staining target, the cells were applied with Rhodamine Phalloidin (1/40, Life Technology) for an hour, rabbit anti-mDia2 antibodies (1/100, Life Technology), rabbit anti-myosin IIa antibodies (1/100, Cell Signaling), or rabbit anti Tpm4 antibodies (1/100, abcam) overnight, followed by application of secondary antibodies anti-rabbit (1/250, Life Technology) for an hour, and DAPI staining (300 nM, Thermal Fisher) for 5 min. It was then mounted with Fluoromount G (Electon Microscopy Sciences. Inc.) and imaged by Nikon Eclipse Ti-E microscope. For heatmap distribution of actin, individual images were stacked and averaged in Matlab with reference to the centroid of circular islands and scaled based on the maximum/minimum intensity of the image. For differential heat map, the intensity difference is calculated by subtraction between treated and untreated cases and scaled based on the maximum/minimum intensity difference of the image.

Differentiation of hMSCs. hMSCs were dual induced by replenishing mixed adipogenic and osteogenic induction medium at 1 on 1 ratio every 3 days. On day 6, nucleus was first stained with 2 µg/mL H33342, followed by Fast Blue staining (Sigma-Aldrich, 85L2-1KT) with protocol provided by manufacturer instruction. In brief, cell was washed in fixation solution, containing two part of citrate working solution and three part of acetone, for 1 min and rinsed in DPBS. Cell was then incubated in staining solution consisted with a capsule of Fast Blue RR Salt and 2 ml of Naphthol AS-MX Phosphate Alkaline solution, 0.25% dissolved in 48 ml DI water. After incubation in room temperature for 30 min, the sample was rinsed with DI water to stop the reaction. For Oil Red O staining, cells were first rinsed with DPBS. Then, 60% isopropanol was applied to the cell for 5 min, before staining with 3 mg/ml Oil Red O (Sigma-Aldrich) for 15 min. Cell was rinsed in PBS to remove the remaining staining agent.

Statistical Analysis. Student’s t test was applied to evaluate the statistical difference. The confidence level was set to be 0.05 for all statistical tests. The statistical significance was symbolized by ns (p > 0.05), * (p ≤ 0.05), ** (p value ≤ 0.01), *** (p value ≤ 0.001), or **** (p value ≤ 0.0001).

Acknowledgments

We thank Department of Infectious Diseases and Public Health, City University of Hong Kong and Chinetek Scientific for sharing the Nikon Eclipse Ti2-E microscope with spinning disk confocal illuminator (LDI-7 Laser Diode Illuminator, 89 North) and the technical support. This study was supported by the following: Hong Kong Research Grant Council (11217820 and N_CityU119/19), Innovation and Technology Commission (ITS/098/20), The Science, Technology and Innovation Commission of Shenzhen Municipality (JCYJ20210324134006017), and City University of Hong Kong (9678242 and 6430620).

Author contributions

H.K.K. and T.H.C. conceptualized. H.K.K investigated and performed experiments. H.K.K and T.H.C analyzed data. W.G.L., S.Y.W. and L.T.C. design experiments. C.F.C., W.G.L., H.H. and J.C.W collect data. H.L.C. constructed model. H.K.K. and T.H.C. wrote the manuscript. Funding acquisition, M.L.L. and T.H.C. Supervision, T.H.C..

Competing interests

The authors declare no competing interests.

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There is NO Competing Interest.

NatureCommunicationSI20220120.docx
Supplementary Materials
SupplementaryMovie1Phasecontrastandnucleusrotationonculturedish.mp4
Supplementary Movie 1
SupplementaryMovie2Controlon2500laser.mp4
Supplementary Movie 2
SupplementaryMovie3Controlon2500fluorescence.mp4
Supplementary Movie 3
SupplementaryMovie4Animationactinpolymerization.mp4
Supplementary Movie 4
SupplementaryMovie5Controlon75013.mp4
Supplementary Movie 5
SupplementaryMovie6LatAon2500Laser.mp4
Supplementary Movie 6
SupplementaryMovie7LatAon2500GFP.mp4
Supplementary Movie 7
SupplementaryMovie8myosinonactin.mp4
Supplementary Movie 8
SupplementaryMovie9myosincontributingtolefthandedmotiononradialfiber.mp4
Supplementary Movie 9

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Cell chirality reversal through tilted balance between polymerization of radial fibers and clockwise-swirling of transverse arc

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